The objective of this proposal is to provide training in the molecular and cellular biology of the liver, and novel microengineering techniques for studying cell-cell interactions. This will be accomplished through experiments that will enhance the understanding of mechanisms underlying fibrosis in nonalcoholic fatty liver disease (NAFLD). The significance of this proposal lies in the high prevalence of NAFLD and the current lack of understanding of the molecular and cellular mechanisms underlying its progression to nonalcoholic steatohepatitis (NASH). NAFLD is estimated to affect 20-25% of the U.S. population and more than 67% of obese persons. Simple fatty liver without inflammation or fibrosis often has a benign, nonprogressive course. Defining the mechanisms involved in the induction and maintenance of progressive NAFLD is thus of critical importance. High mobility group box 1 (HMGB1) is a protein secreted by macrophages and monocytes, in response to proinflammatory stimuli. The hypotheses of this proposal are: (1) HMGB1, secreted by Kupffer cells (KCs), activates quiescent hepatic stellate cells (HSCs) by binding the receptor for advanced glycation end products (RAGE); and (2) RAGE ligation induces a RAGE positive-feedback loop that promotes fibrosis and inhibits apoptosis in activated HSCs. The specific aims are: (1) To quantify serum and liver tissue levels of HMGB1 in normal, steatotic, and steatohepatitic rats in vivo, and to evaluate the potential of KCs isolated from each of these animal models to secrete HMGB1 in response to cytokine stimulation in vitro; (2) To characterize the role of HMGB1 in KC-mediated HSC activation, and inhibition of apoptosis, in vitro and in vivo; (3) to evaluate the effects of HMGB1-RAGE blockade on the pathogenesis and progression of steatohepatitis in vivo.